Methods for biosynthesizing methacrylate

Abstract

This document describes biochemical pathways for producing methacrylate from precursors such as pyruvate via isobutyraldehyde and isobutyryl-CoA, using enzymes such as one or more thioesterases, transferases, or dehydrogenases, as well as recombinant hosts expressing one or more of such enzymes.

What is claimed is: 
     
         1 . A method of producing methacrylate, said method comprising enzymatically converting methacryloyl-CoA to methacrylate using a thioesterase or CoA-transferase. 
     
     
         2 . The method of  claim 1 , wherein said thioesterase is classified under EC 3.1.2.-. 
     
     
         3 . The method of  claim 1 , wherein said thioesterase is the gene product of tesB or YciA. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. 
     
     
         5 . The method of  claim 1 , wherein said CoA-transferase is classified under EC 2.8.3.-. 
     
     
         6 . The method of  claim 5 , wherein said CoA-transferase has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. 
     
     
         7 . The method of any one of  claims 1 - 5 , wherein methacryloyl-CoA is enzymatically synthesized from pyruvate. 
     
     
         8 . The method of  claim 7 , wherein methacryloyl-CoA is enzymatically synthesized from pyruvate via isobutyraldehyde. 
     
     
         9 . The method of  claim 8 , wherein methacryloyl-CoA is enzymatically synthesized from pyruvate using one or more of the following enzymes: an acetolactate synthase; a dihydroxyisovalerate dehydrogenase; a 2,3-dihydroxyisovalerate dehydratase; a 2-oxovalerate decarboxylase; a phenylacetaldehyde dehydrogenase, isobutyraldehyde dehydrogenase or a lactaldehyde dehydrogenase; or a CoA transferase or a ligase; or an acyl-CoA dehydrogenase. 
     
     
         10 . The method of  claim 7 , wherein methacryloyl-CoA is enzymatically synthesized from pyruvate via conversion of 2-oxo-isovalerate to isobutyryl-CoA. 
     
     
         11 . The method of  claim 10 , wherein methacryloyl-CoA is enzymatically synthesized from pyruvate using one or more of the following enzymes: an acetolactate synthase; a dihydroxyisovalerate dehydrogenase; a 2,3-dihydroxyisovalerate dehydratase; a branch chain dehydrogenase; or an acyl-CoA dehydrogenase. 
     
     
         12 . A method of producing methacrylate, said method comprising enzymatically synthesizing isobutyraldehyde from pyruvate, and enzymatically converting isobutryaldehyde to methacrylate. 
     
     
         13 . The method of  claim 12 , wherein isobutryaldehyde is converted to isobutyrate using a phenylacetaldehyde dehydrogenase, isobutyraldehyde dehydrogenase, or lactaldehyde dehydrogenase, isobutyrate is converted to isobutyryl-CoA using a CoA transferase or a ligase, isobutyryl-CoA is converted to methacryloyl-CoA using an acyl-CoA dehydrogenase, and methacryloyl-CoA is converted to methacrylic acid using a thioesterase or CoA transferase. 
     
     
         14 . The method of  claim 13 , wherein said phenylacetaldehyde dehydrogenase is classified under EC 1.2.1.39. 
     
     
         15 . The method of  claim 14 , wherein said phenylacetaldehyde dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. 
     
     
         16 . The method of  claim 13 , wherein said isobutyraldehyde dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. 
     
     
         17 . The method of  claim 13 , wherein said CoA transferase is a propionate CoA transferase classified under EC 2.8.3.1. 
     
     
         18 . The method of  claim 17 , wherein said propionate CoA-transferase has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. 
     
     
         19 . The method of  claim 13 , wherein said ligase is a propionate CoA ligase classified under EC 6.2.1.13. 
     
     
         20 . The method of any one of  claims 13 - 19 , wherein said acyl-CoA dehydrogenase is an isobutyryl-CoA dehydrogenase. 
     
     
         21 . The method of  claim 20 , wherein said isobutyryl-CoA dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9. 
     
     
         22 . The method of any of the preceding claims, wherein said method is performed in a recombinant host. 
     
     
         23 . The method of  claim 22 , wherein said host is subjected to a cultivation strategy under anaerobic, aerobic or micro-aerobic cultivation conditions. 
     
     
         24 . The method of  claim 22  or  23 , wherein said host is cultured under conditions of nutrient limitation either via nitrogen, phosphate or oxygen limitation. 
     
     
         25 . The method according to  claim 22  or  23 , wherein said recombinant host cells are retained using ceramic hollow fiber membranes to maintain a high cell density during fermentation. 
     
     
         26 . The method of  claim 22  or  23 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         27 . The method of  claim 26 , wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin such as levulinic acid and furfural, lignin, triglycerides such as glycerol and fatty acids, agricultural waste or municipal waste. 
     
     
         28 . The method of  claim 26 , wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, non-volatile residue (NVR) or caustic wash waste stream from cyclohexane oxidation processes. 
     
     
         29 . The method of  claim 22 , wherein the host is a prokaryote or a eukaryote. 
     
     
         30 . The method of  claim 29 , wherein the host's tolerance to high concentrations of methacrylic acid is improved through continuous cultivation in a selective environment. 
     
     
         31 . The method of  claim 29 , wherein endogenous degradation pathways of central metabolites and central precursors leading to and including methacrylic acid are attenuated in the host. 
     
     
         32 . The method of  claim 22 , wherein the efflux of methacrylic acid across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for methacrylic acid. 
     
     
         33 . A recombinant host comprising at least one exogenous nucleic acid encoding a phenylacetaldehyde dehydrogenase, wherein said recombinant host produces methacrylic acid. 
     
     
         34 . The host of  claim 33 , wherein said phenylacetaldehyde dehydrogenase is classified under EC 1.2.1.39. 
     
     
         35 . The host of  claim 33  or  claim 34 , wherein said phenylacetaldehyde dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. 
     
     
         36 . The host of any one of  claims 33 - 35 , said host further comprising an exogenous propionate CoA transferase. 
     
     
         37 . The host of  claim 36 , wherein said propionate CoA transferase has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. 
     
     
         38 . The host of any one of  claims 33 - 35 , said host further comprising an exogenous propionate CoA ligase. 
     
     
         39 . The host of any one of  claims 33 - 38 , said host further comprising an exogenous thioesterase. 
     
     
         40 . The host of  claim 39 , wherein said thioesterase is the gene product of YciA or tesB. 
     
     
         41 . The host of  claim 39 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. 
     
     
         42 . The host of any one of  claims 33 - 41 , said host further comprising an exogenous isobutyryl-CoA dehydrogenase. 
     
     
         43 . The host of  claim 42 , wherein said isobutyryl-CoA dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:9.